U.S. patent application number 16/307643 was filed with the patent office on 2019-08-29 for treatments of non-alcoholic steatohepatitis (nash).
The applicant listed for this patent is DEUTSCHES KREBSFORSCHUNGSZENTRUM STIFTUNG DES OFFENTLICHEN RECHTS. Invention is credited to Mathias HEIKENWALDER, Achim WEBER, Lars ZENDER.
Application Number | 20190262379 16/307643 |
Document ID | / |
Family ID | 56296569 |
Filed Date | 2019-08-29 |
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United States Patent
Application |
20190262379 |
Kind Code |
A1 |
HEIKENWALDER; Mathias ; et
al. |
August 29, 2019 |
TREATMENTS OF NON-ALCOHOLIC STEATOHEPATITIS (NASH)
Abstract
The present invention provides novel compounds that target
thrombocyte activity or aggregation capacity through cellular
components for the treatment of diseases associated with
Non-Alcoholic Fatty Liver Disease (NAFLD). The invention provides
these compounds for treating non-alcoholic steatohepatitis (NASH),
a progressed stage of NAFL (non-alcoholic fatty liver), in order to
avoid the development of liver cirrhosis and Hepatocellular
Carcinoma (HCC). Further provided are pharmaceutical compositions,
comprising the compounds of the invention, and methods for
screening new NASH therapeuticals.
Inventors: |
HEIKENWALDER; Mathias;
(Heidelberg, DE) ; ZENDER; Lars; (ROTTENBURG,
DE) ; WEBER; Achim; (ZURICH, CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DEUTSCHES KREBSFORSCHUNGSZENTRUM STIFTUNG DES OFFENTLICHEN
RECHTS |
HEIDELBERG |
|
DE |
|
|
Family ID: |
56296569 |
Appl. No.: |
16/307643 |
Filed: |
June 28, 2017 |
PCT Filed: |
June 28, 2017 |
PCT NO: |
PCT/EP2017/066025 |
371 Date: |
December 6, 2018 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/616 20130101;
A61K 9/0019 20130101; A61K 9/0053 20130101; A61P 1/16 20180101;
C12N 15/113 20130101; A61K 31/7105 20130101; C12N 2310/11 20130101;
A61K 31/00 20130101; A61K 31/192 20130101; A61K 31/713 20130101;
C12N 2310/20 20170501; A61K 31/405 20130101; A61K 9/0014
20130101 |
International
Class: |
A61K 31/7105 20060101
A61K031/7105; A61P 1/16 20060101 A61P001/16; A61K 31/616 20060101
A61K031/616; A61K 31/192 20060101 A61K031/192; A61K 31/405 20060101
A61K031/405; C12N 15/113 20060101 C12N015/113; A61K 9/00 20060101
A61K009/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 28, 2016 |
EP |
16176755.3 |
Claims
1. A method for the treatment or prevention of non-alcoholic
steatopepatitis (NASH), wherein said method comprises
administering, to a subject in need of such treatment, an inhibitor
of thrombocyte aggregation or activation.
2. The method according to claim 1, wherein the inhibitor is an
inhibitor of Gp1b, GpV, GpIX, Factor 8, or Nbeal2.
3. The method according to claim 1, wherein the inhibitor is an
antisense nucleic acid, CRISPR-Cas9 like gene editing construct, an
antibody or an antigen binding fragment thereof, a small molecule,
a peptide, a ribozyme, or a recombinant protein.
4. The method according to claim 3, wherein the antisense nucleic
acid is an RNA interference inducing nucleic acid.
5. The method according to claim 3, wherein an antibody, or antigen
binding fragment thereof, is an anti-Gp1b antibody, an anti-GpV
antibody, an anti-GpIX antibody, an anit-Factor 8 antibody, or an
anti-Nbeal2 antibody that specifically and selectively binds to a
GpV, GpIX, Factor 8, or Nbeal2 protein and inhibits its
function.
6. The method according to claim 1, wherein the treatment is an
alleviation or a reduced progression of NASH.
7. The method according to claim 1, wherein the treatment is a
reduced, stalled, or reversed progression of NASH into liver
cirrhosis.
8. The method according to claim 1, wherein the treatment is a
prevention of HCC in a NASH-patient at risk to develop cirrhosis
and/or HCC.
9. The method according to claim 1, wherein the treatment is
performed in a patient at risk of developing NASH, wherein the
patient is a diabetic patient, an obese patient, or a patient
suffering from metabolic syndrome or from another metabolic
disorder.
10. The method according to claim 1, wherein the subject to be
treated does not have a condition selected from the group
consisting of alcoholic liver injury, drug-induced liver injury,
chronic active hepatitis, hepatic steatosis and hepatocyte
apoptosis.
11. A pharmaceutical composition for use in the treatment or
prevention of NASH, comprising an inhibitor of thrombocytes and a
pharmaceutically acceptable carrier and/or excipient.
12. A method for identifying whether a compound has an activity for
treatment of NASH, the method comprising contacting a cell
expressing a protein selected from the group consisting of Gp1b,
GpV, GpIX, Factor 8, and Nbeal2, with a candidate compound,
determining the expression, stability and/or activity of said
protein compared to a control cell expressing the protein and which
is not contacted with the candidate compound, wherein a reduced
expression, stability and/or activity of the protein upon
contacting with the candidate compound indicates that the candidate
compound has an activity for treatment of NASH.
13. The method according to claim 12, wherein the candidate
compound is an antisense nucleic acid, CRISPR-Cas9 like gene
editing construct, an antibody or an antigen binding fragment
thereof, a viral construct, a small molecule, a peptide, a
ribozyme, or a recombinant protein.
14. The method according to claim 3, wherein the antisense nucleic
acid is an RNA interference inducing nucleic acid comprising a
sequence complementary to a Gp1b mRNA sequence.
15. The method according to claim 3, wherein the antisense nucleic
acid is a siRNA, shRNA, miRNA, or LNA-construct.
16. The method according to claim 1, wherein the treatment is a
reduced, stalled, or reversed progression of NASH into
hepatocellular carcinoma (HCC).
17. The method, according to claim 10, wherein the patient suffers
from an inflamed fatty liver.
Description
FIELD OF THE INVENTION
[0001] The present invention provides novel compounds that target
thrombocyte activity or aggregation capacity through cellular
components for the treatment of diseases associated with
Non-Alcoholic Fatty Liver Disease (NAFLD). The invention provides
these compounds for treating non-alcoholic steatohepatitis (NASH),
a progressed stage of NAFL (non-alcoholic fatty liver), in order to
avoid the development of liver cirrhosis and Hepatocellular
Carcinoma (HCC). Further provided are pharmaceutical compositions,
comprising the compounds of the invention, and methods for
screening new NASH therapeuticals.
DESCRIPTION
[0002] Changes in life-style over the last decades such as high
caloric intake (e.g. through high fat, high fructose and high
glucose diet) combined with sedentary life style have increased the
incidence of overweight and metabolic syndrome, which is
characterized by abdominal obesity, insulin resistance, hypertonia
and dyslipidemia. The latest WHO cancer report predicts a doubling
in cancer incidence within the next two decades, whereof the great
majority will be attributable to modifiable risk factors such as
high caloric intake, smoking and sedentary lifestyle (Stewart and
Wild, 2014). A strong link between obesity and cancer incidence is
well-established and a body mass index (BMI) >25 substantially
increases the risk to develop several cancers (Calle and Kaaks,
2004). The liver, which is the most important metabolic organ of
the body, is strongly affected by a chronic state of hypercaloric
uptake, overweight, sedentary lifestyle and the resulting pathology
(metabolic syndrome).
[0003] Non-alcoholic fatty liver disease (NAFLD), comprising
several liver diseases including NAFL and NASH, which is the most
frequent liver disease world-wide, is a clinical manifestation of
overweight and metabolic syndrome. NAFL is a chronic disease that
can last several decades and is characterized by predominant
macrovesicular steatosis of the liver. The prevalence of NAFL is
increasing globally (Loomba et al., 2013). Currently, 90 million
Americans and 40 million Europeans suffer from NAFLD.
Interestingly, also developing countries show a strong rise in
NAFLD cases, reflecting the consequences of industrialization and a
concomitant "Western life style". A significant number of NAFL
patients develop non-alcoholic steatohepatitis (NASH), fibrosis and
subsequently hepatocellular carcinoma (HCC). At the same time, the
amount of people suffering from NASH is increasing in the USA and
Europe. Consequently, obesity, steatosis and steatohepatitis have
attracted increased attention due to rising HCC incidence in
Western countries (White et al., 2012). In line, the most common
etiology of HCC in industrialized countries is currently switching
from chronic viral infections (e.g. Hepatitis B and C viruses) to
obesity, making HCC the most rapidly increasing type of cancer in
the U.S., with a similar trend observed in Europe
(AmericanCancerSociety, 2007).
[0004] Today, a detailed understanding of how chronic steatosis
develops into NASH and what factors control NASH to HCC transition
is still lacking. At the same time no efficient therapeutics exist
to treat NASH and treatment options for late stage HCC are limited,
prolonging the life span of patients for only 3 to 6 months
(Villanueva et al., 2014). It becomes increasingly clear that a
number of pathways are involved in the pathogenesis of NASH and its
progression to advanced stages of liver disease. These pathways may
vary in different cohorts of patients with NASH. Understanding
which pathways play a role in the development of NASH and NASH
driven HCC will be critical before launching treatment modalities.
Indeed, NASH is associated with metabolic syndrome in most cases in
the Western world, but can manifest also at a lower BMI, e.g. in
Asian countries and many patients do not seem to have insulin
resistance. These observations suggest that additional genetic
factors are potentially involved in disease progression (Loomba et
al., 2013).
[0005] Clearly there remains a significant unmet need for novel
therapeutic approaches to target NASH progression into cirrhosis
and HCC. The object of the present invention is therefore to
provide novel treatment targets to this end.
[0006] The above problem is solved by altering the activation and
aggregation potential of platelets in order to inhibit inflammation
in the liver as well as their deleterious effect on hepatocyte
metabolism in the context of a high calorie diet. The invention
therefore provides in a first aspect an inhibitor of thrombocytes
for use in the treatment or prevention of a non-alcoholic fatty
liver disease (NAFLD).
[0007] A thrombocyte inhibitor in context of the invention is also
referred to as an antiplatelet drug or platelet aggregation
inhibitor, and may be selected from any agent known in the art to
inhibit the activity, proliferation or aggregation of thrombocytes.
Examples of such compounds are agents that inhibit platelet
function by inhibiting the aggregation, or by adhesion or granular
secretion of platelets. Anti-platelet agents used in context of the
invention include, but are not limited to, the various known
non-steroidal anti-inflammatory drugs (NSAIDS) such as aspirin,
ibuprofen, naproxen, sulindac, indomethacin, mefenamate, droxicam,
diclofenac, sulfinpyrazone, piroxicam, and pharmaceutically
acceptable salts or prodrugs thereof. In another embodiment, the
anti-platelet agent is an Ilb/IIIa antagonists (e.g., tirofiban,
eptifibatide, and abciximab), thromboxane-A2-receptor antagonists
(e.g., ifetroban), thromboxane-A2-synthetase inhibitors, PDE-III
inhibitors (e.g., dipyridamole), and pharmaceutically acceptable
salts or prodrugs thereof. In another embodiment, the term
anti-platelet agents (or platelet inhibitory agents), refers to ADP
(adenosine diphosphate) receptor antagonists, which is in one
embodiment, an antagonists of the purinergic receptors P.sub.2 Yi
and P.sub.2 Yn--In one embodiment, P.sub.2 Yi.sub.2 receptor
antagonists is ticlopidine, clopidogrel, or their combination and
pharmaceutically acceptable salts or prodrugs thereof.
[0008] In some preferred embodiments of the invention, there is
provided an inhibitor of inhibitor of Gp1b, GPV, GPIX, Factor 8, or
Nbeal2 for use in the treatment or prevention of a non-alcoholic
fatty liver disease (NAFLD). The above protein denominations are
used for the following protein names in parenthesis GpIb
(Glycoprotein Ib), GpV (Glycoprotein V), GPIX (Glycoprotein IX),
Factor 8 (or Factor VIII; FVIII), and Nbeal2 (Neurobeachin-like
protein 2).
[0009] An "inhibitor of Gp1b, GPV, GPIX, Factor 8, or Nbeal2" is an
antagonist of a mammalian homologue of Gp1b, GPV, GPIX, Factor 8,
or Nbeal2 respectively, preferably human Gp1b, GPV, GPIX, Factor 8,
or Nbeal2. As used herein, the term "inhibitor of Gp1b, GPV, GPIX,
Factor 8, or Nbeal2" means a substance that affects a decrease in
the amount or rate of Gp1b, GPV, GPIX, Factor 8, or Nbeal2
expression or activity. Such a substance can act directly, for
example, by binding to Gp1b, GPV, GPIX, Factor 8, or Nbeal2 and
decreasing the amount or rate of Gp1b, GPV, GPIX, Factor 8, or
Nbeal2 expression or activity. A Gp1b, GPV, GPIX, Factor 8, or
Nbeal2-antagonist can also decrease the amount or rate of Gp1b,
GPV, GPIX, Factor 8, or Nbeal2 expression or activity, for example,
by binding to Gp1b, GPV, GPIX, Factor 8, or Nbeal2 in such a way as
to reduce or prevent interaction of Gp1b, GPV, GPIX, Factor 8, or
Nbeal2 with a ligand; by binding to Gp1b, GPV, GPIX, Factor 8, or
Nbeal2 and modifying it, such as by removal or addition of a
moiety; and by binding to Gp1b, GPV, GPIX, Factor 8, or Nbeal2 and
reducing its stability. A Gp1b, GPV, GPIX, Factor 8, or
Nbeal2-antagonist can also act indirectly, for example, by binding
to a regulatory molecule or gene region so as to modulate
regulatory protein or gene region function and affect a decrease in
the amount or rate of Gp1b, GpV, GpIX, Factor 8, or Nbeal2
expression or activity. Thus, a Gp1b, GpV, GpIX, Factor 8, or
Nbeal2-antagonist can act by any mechanisms that result in decrease
in the amount or rate of Gp1b, GpV, GpIX, Factor 8, or Nbeal2
expression, stability or activity.
[0010] A Gp1b, GpV, GpIX, Factor 8, or Nbeal2-antagonist can be,
for example, a naturally or non-naturally occurring macromolecule,
such as a polypeptide, peptide, peptidomimetic, nucleic acid,
carbohydrate or lipid. A Gp1b, GpV, GpIX, Factor 8, or
Nbeal2-antagonist further can be an antibody, or antigen-binding
fragment thereof, such as a mono-clonal antibody, humanized
antibody, chimeric antibody, minibody, bifunctional antibody,
single chain antibody (scFv), variable region fragment (Fv or Fd),
Fab or F(ab)2. A Gp1b, GpV, GpIX, Factor 8, or Nbeal2-antagonist
can also be polyclonal antibodies specific for Gp1b, GpV, GpIX,
Factor 8, or Nbeal2. A Gp1b, GpV, GpIX, Factor 8, or
Nbeal2-antagonist further can be a partially or completely
synthetic derivative, analog or mimetic of a naturally occurring
macromolecule, or a small organic or inorganic molecule.
[0011] A Gp1b, GpV, GpIX, Factor 8, or Nbeal2-antagonist that is an
antibody can be, for example, an antibody that binds to Gp1b, GpV,
GpIX, Factor 8, or Nbeal2 and inhibits binding to a Gp1b, GpV,
GpIX, Factor 8, or Nbeal2 ligand, or alters the activity of a
molecule that regulates Gp1b, GpV, GpIX, Factor 8, or Nbeal2
expression or activity, such that the amount or rate of Gp1b, GpV,
GpIX, Factor 8, or Nbeal2 expression or activity is decreased. An
antibody useful in a method of the invention can be a naturally
occurring antibody, including a monoclonal or polyclonal antibodies
or fragment thereof, or a non-naturally occurring antibody,
including but not limited to a single chain antibody, chimeric
antibody, bifunctional antibody, complementarity determining
region-grafted (CDR-grafted) antibody and humanized antibody or an
antigen-binding fragment thereof.
[0012] A Gp1b, GpV, GpIX, Factor 8, or Nbeal2-antagonist that is a
nucleic acid can be, for example, an anti-sense nucleotide
sequence, an RNA, DNA, RNA/DNA, or LNA molecule, or an aptamer
sequence. An anti-sense nucleotide sequence can bind to a
nucleotide sequence within a cell and modulate the level of
expression of Gp1b, GpV, GpIX, Factor 8, or Nbeal2, respectively,
or modulate expression of another gene that controls the expression
or activity of Gp1b, GpV, GpIX, Factor 8, or Nbeal2. Similarly, an
RNA molecule, such as a catalytic ribozyme, can bind to and alter
the expression of a Gp1b, GpV, GpIX, Factor 8, or Nbeal2 gene, or
other gene that controls the expression or activity of Gp1b, GpV,
GpIX, Factor 8, or Nbeal2. An aptamer is a nucleic acid sequence
that has a three dimensional structure capable of binding to a
molecular target.
[0013] A Gp1b, GpV, GpIX, Factor 8, or Nbeal2-antagonist that is a
nucleic acid also can be a double-stranded RNA molecule for use in
RNA interference methods. RNA interference (RNAi) is a process of
sequence-specific gene silencing by post-transcriptional RNA
degradation, which is initiated by double-stranded RNA (dsRNA)
homologous in sequence to the silenced gene. A suitable
double-stranded RNA (dsRNA) for RNAi contains sense and antisense
strands of about 21 contiguous nucleotides corresponding to the
gene to be targeted that form 19 RNA base pairs, leaving overhangs
of two nucleotides at each 3' end (Elbashir et al., Nature
411:494-498 (2001); Bass, Nature 411:428-429 (2001); Zamore, Nat.
Struct. Biol. 8:746-750 (2001)). dsRNAs of about 25-30 nucleotides
have also been used successfully for RNAi (Karabinos et al., Proc.
Natl. Acad. Sci. USA 98:7863-7868 (2001). dsRNA can be synthesized
in vitro and introduced into a cell by methods known in the art. As
already indicated, the antisense nucleic acid is an RNA
interference inducing nucleic acid, preferably comprising a
sequence complementary to a Gp1b, GpV, GpIX, Factor 8, or Nbeal2
mRNA sequence, such as, but not limited to, a siRNA, shRNA, miRNA,
LNA-constructs. Such constructs are known in the art and easy to
prepare by the skilled artisan.
[0014] Another option is furthermore the use of CRISPR/Cas9 or
similar gene editing approaches to introduce mutations into the
genes of Gp1b, GpV, GpIX, Factor 8, or Nbeal2. Such CRISPR gene
editing constructs shall therefore be comprised by the term Gp1b,
GpV, GpIX, Factor 8, or Nbeal2--antagonist insofar they are used to
impair expression, stability or function of Gp1b, GpV, GpIX, Factor
8, or Nbeal2.
[0015] Preferred embodiments of the present invention pertain to
non-alcoholic steatohepatitis (NASH) as NAFLD of the invention.
Therefore, it is preferred in context of the invention that the
treatment of NAFLD involves a patient group suffering from NASH and
not NAFL, and therefore patients which already developed the more
advanced form of a NAFLD. Therefore, the patient group preferable
to be treated with the inhibitors of the invention are patients
showing signs of fat and inflammation, sometimes also damage, in
the liver. In some embodiments the patients to be treated in
context of the invention are patients which do not suffer from
signs of cirrhosis or hepatocellular carcinoma (HCC). Other
embodiments however provide that patients to be treated in
accordance with the invention already show signs of cirrhosis
and/or HCC. Preferably the inhibitors of the invention are for use
in a treatment which is a prevention of HCC in a NASH-patient at
risk to develop cirrhosis and/or HCC.
[0016] A patient at risk of developing NAFLD (preferably NASH) in
context of the invention is a preferred subject to benefit for the
inhibitors for use of the invention. Such a risk patient is for
example a diabetic patient, an obese patient, or a patient
suffering from the metabolic syndrome or another metabolic
disorder.
[0017] Most preferably the subject to be treated in context of the
invention (patient group) does not have a condition selected from
the following group consisting of alcoholic liver injury,
drug-induced liver injury, chronic active hepatitis, cirrhosis,
liver cancer, hepatic steatosis and hepatocyte apoptosis.
[0018] The present invention provides for alleviation or a reduced
progression of the NAFLD to be treated, in particular NASH.
Therefore, the invention provides a treatment of NAFLD preferably
NASH. The inhibitors of the invention are preferably used to
reduce, stall, or reverse progression of NASH into liver cirrhosis,
and/or progression of NASH into hepatocellular carcinoma (HCC).
Thus treatment in context of the invention is preferably an
alleviation of NASH into a non-NASH state of NAFLD.
[0019] In another aspect of the invention there is provided a
pharmaceutical composition for use in the treatment or prevention
of a non-alcoholic fatty liver disease (NAFLD), as described herein
above, the pharmaceutical composition comprising an inhibitor of
thrombocytes or an inhibitor of Gp1b, GpV, GpIX, Factor 8, or
Nbeal2 (as described herein above) and a pharmaceutically
acceptable carrier and/or excipient.
[0020] As used herein the language "pharmaceutically acceptable
carrier" is intended to include any and all solvents, solubilizers,
fillers, stabilizers, binders, absorbents, bases, buffering agents,
lubricants, controlled release vehicles, diluents, emulsifying
agents, humectants, lubricants, dispersion media, coatings,
antibacterial or antifungal agents, isotonic and absorption
delaying agents, and the like, compatible with pharmaceutical
administration. The use of such media and agents for
pharmaceutically active substances is well-known in the art. Except
insofar as any conventional media or agent is incompatible with the
active compound, use thereof in the compositions is contemplated.
Supplementary agents can also be incorporated into the
compositions. In certain embodiments, the pharmaceutically
acceptable carrier comprises serum albumin.
[0021] The pharmaceutical composition of the invention is
formulated to be compatible with its intended route of
administration. Examples of routes of administration include
parenteral, e.g., intrathecal, intra-arterial, intravenous,
intradermal, subcutaneous, oral, transdermal (topical) and
transmucosal administration. Most preferably the route of
administration is a route that directly targets the liver of a
subject to be treated.
[0022] In therapeutic applications, compositions are administered
to a patient already suffering from a NAFLD, as described, in an
amount sufficient to cure or at least partially stop the symptoms
of the disease and its complications. An appropriate dosage of the
pharmaceutical composition is readily determined according to any
one of several well-established protocols. For example, animal
studies (for example on mice or rats) are commonly used to
determine the maximal tolerable dose of the bioactive agent per
kilogram of weight. In general, at least one of the animal species
tested is mammalian. The results from the animal studies can be
extrapolated to determine doses for use in other species, such as
humans for example. What constitutes an effective dose also depends
on the nature and severity of the disease or condition, and on the
general state of the patient's health.
[0023] In prophylactic applications, compositions containing, for
example Gp1b, GpV, GpIX, Factor 8, or Nbeal2 antagonists, are
administered to a patient susceptible to or otherwise at risk of a
hepatic disease. Such an amount is defined to be a
"prophylactically effective" amount or dose. In this use, the
precise amount depends on the patient's state of health and
weight.
[0024] In both therapeutic and prophylactic treatments, the
antagonist contained in the pharmaceutical composition can be
administered in several dosages or as a single dose until a desired
response has been achieved. The treatment is typically monitored
and repeated dosages can be administered as necessary. Compounds of
the invention may be administered according to dosage regimens
established whenever inactivation of p1b, GpV, GpIX, Factor 8, or
Nbeal2 is required.
[0025] The daily dosage of the products may be varied over a wide
range from 0.01 to 1,000 mg per adult per day. Preferably, the
compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0,
15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for
the symptomatic adjustment of the dosage to the patient to be
treated. A medicament typically contains from about 0.01 mg to
about 500 mg of the active ingredient, preferably from 1 mg to
about 100 mg of the active ingredient. An effective amount of the
drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to
about 20 mg/kg of body weight per day, especially from about 0.001
mg/kg to 10 mg/kg of body weight per day. It will be understood,
however, that the specific dose level and frequency of dosage for
any particular patient may be varied and will depend upon a variety
of factors including the activity of the specific compound
employed, the metabolic stability, and length of action of that
compound, the age, the body weight, general health, sex, diet, mode
and time of administration, rate of excretion, drug combination,
the severity of the particular condition, and the host undergoing
therapy.
[0026] In the pharmaceutical compositions of the present invention
for oral, sublingual, subcutaneous, intramuscular, intravenous,
transdermal, local or rectal administration, the active principle,
alone or in combination with another active principle, can be
administered in a unit administration form, as a mixture with
conventional pharmaceutical supports, to animals and human beings.
Suitable unit administration forms comprise oral-route forms such
as tablets, gel capsules, powders, granules and oral suspensions or
solutions, sublingual and buccal administration forms, aerosols,
implants, subcutaneous, transdermal, topical, intraperitoneal,
intramuscular, intravenous, subdermal, transdermal, intrathecal and
intranasal administration forms and rectal administration
forms.
[0027] The appropriate unit forms of administration include forms
for oral administration, such as tablets, gelatine capsules,
powders, granules and solutions or suspensions to be taken orally,
forms for sublingual and buccal administration, aerosols, implants,
forms for subcutaneous, intramuscular, intravenous, intranasal or
intraocular administration and forms for rectal administration.
[0028] In the pharmaceutical compositions of the present invention,
the active principle is generally formulated as dosage units
containing from 0.5 to 1000 mg, preferably from 1 to 500 mg, more
preferably from 2 to 200 mg of said active principle per dosage
unit for daily administrations.
[0029] When preparing a solid composition in the form of tablets, a
wetting agent such as sodium laurylsulfate can be added to the
active principle optionally micronized, which is then mixed with a
pharmaceutical vehicle such as silica, gelatine, starch, lactose,
magnesium stearate, talc, gum arabic or the like. The tablets can
be coated with sucrose, with various polymers or other appropriate
substances or else they can be treated so as to have a prolonged or
delayed activity and so as to release a predetermined amount of
active principle continuously.
[0030] A preparation in the form of gelatin capsules is obtained by
mixing the active principle with a diluent such as a glycol or a
glycerol ester and pouring the mixture obtained into soft or hard
gelatine capsules.
[0031] A preparation in the form of a syrup or elixir can contain
the active principle together with a sweetener, which is preferably
calorie-free, methyl-paraben and propylparaben as an antiseptic, a
flavoring and an appropriate color.
[0032] The water-dispersible powders or granules can contain the
active principle mixed with dispersants or wetting agents, or
suspending agents such as polyvinylpyrrolidone, and also with
sweeteners or taste correctors.
[0033] Rectal administration is effected using suppositories
prepared with binders which melt at the rectal temperature, for
example cacao butter or polyethylene glycols.
[0034] Parenteral, intranasal or intraocular administration is
effected using aqueous suspensions, isotonic saline solutions or
sterile and injectable solutions which contain pharmacologically
compatible dispersants and/or wetting agents, for example propylene
glycol, butylene glycol, or polyethylene glycol.
[0035] Thus a co-solvent, for example an alcohol such as ethanol or
a glycol such as polyethylene glycol or propylene glycol, and a
hydrophilic surfactant such as Tween..RTM. 80, can be used to
prepare an aqueous solution injectable by intravenous route. The
active principle can be solubilized by a triglyceride or a glycerol
ester to prepare an oily solution injectable by intramuscular
route.
[0036] Transdermal administration is effected using multilaminated
patches or reservoirs into which the active principle is in the
form of an alcoholic solution.
[0037] Administration by inhalation is effected using an aerosol
containing for example sorbitan trioleate or oleic acid together
with trichlorofluoromethane, dichlorotetrafluoroethane or any other
biologically compatible propellant gas.
[0038] The active principle can also be formulated as microcapsules
or microspheres, optionally with one or more carriers or
additives.
[0039] Among the prolonged-release forms which are useful in the
case of chronic treatments, implants can be used. These can be
prepared in the form of an oily suspension or in the form of a
suspension of microspheres in an isotonic medium.
[0040] The above problem is furthermore solved by a method for
identifying whether a compound has an activity towards a treatment
of NAFLD, preferably NASH (and referring to the particular medical
indications as described above) the method comprising contacting a
cell expressing a protein selected from the group consisting of
Gp1b, GpV, GpIX, Factor 8, or Nbeal2, with a candidate compound,
determining the expression, stability and/or activity of said
protein compared to a control cell expressing the protein and which
is not contacted with the candidate compound, wherein a reduced
expression, stability and/or activity of the protein upon
contacting with the candidate compound indicates that the candidate
compound has an activity towards a treatment of NAFLD.
[0041] The candidate compound is preferably selected from antisense
nucleic acid, CRISPR-Cas9 like gene editing construct, an antibody,
or an antigen binding fragment thereof, a viral construct, a small
molecule, a peptide, a ribozyme, or a recombinant protein. In
general, for the screening method of the invention any potential
inhibitor of Gp1b, GpV, GpIX, Factor 8, or Nbeal2 as defined
herein, may be used as a candidate compound.
[0042] The present invention will now be further described in the
following examples with reference to the accompanying figures and
sequences, nevertheless, without being limited thereto. For the
purposes of the present invention, all references as cited herein
are incorporated by reference in their entireties. In the
Figures:
[0043] FIG. 1: Cholesterol Diet--High Fat Diet (CD-HFD) in mice
recapitulates human NASH and NASH triggered HCC pathology
[0044] FIG. 2: CD-HFD recapitulates the inflammatory profile of
human NASH.
[0045] FIG. 3: Increase of platelets and aggregation as a sign of
activation in the liver of mice suffering from NASH upon 6 months
CD-HFD
[0046] FIG. 4: Analysis of activation and accumulation capacity of
platelets analysed by flow cytometry from peripheral blood
[0047] FIG. 5: Aspirin/Clopidrogrel (Asp/Clop), a thrombocyte
aggregation inhibitor, treatment reduces the capacity of platelets
aggregate and prevents NASH and aberrant hepatic lipid
metabolism
[0048] FIG. 6: Reduced amount of intrahepatic immune cells in
Aps/Clp treated mice FIG. 7: Inhibiting the activity/aggregation of
platelets reduces the number of HCC
[0049] FIG. 8: COX1, COX2 inhibitor does not block steatosis or
NASH development upon CD-HFD
[0050] FIG. 9: Ticagrelor blocks NASH and HCC development
[0051] FIG. 10: Nbeal2-/- mice lack development of markers
indicative of NASH development
[0052] FIG. 11: GP1b-/- mice lack steatosis and NASH
development
[0053] FIG. 12: Anti-platelet therapy in humans treats NASH and
HCC
[0054] FIG. 13: Anti-platelet therapy reduces platelet
activation/aggregation, cytokine release and platelet/immune cells
interaction. (a) The level of ADP in the liver extract of mice
under ND, CD-HFD and CD-HFD/Asp-Clo. (data were pooled from two
different independent experiments (n=10 mice per each group).
*P<0.05, **P<0.01, Student's t-test. (b) Prothrombin measured
by enzyme-linked immunosorbent assay (ELISA) in mouse liver extract
extracts from different groups (representative of two independent
experiments (n=5 mice per group). **P<0.01 determined by
Student's t-test. (c) Analysis of cytokines in the liver extracts.
Normalized amount of liver protein extracts were analyzed in ND,
CD-HFD and Asp-Clo treated CD-HFD fed mice. (data are pooled from
two independent experiments, n>10 per each group). Significance
was determined by Student's t-test, *P<0.05,**P<0.01,
***P<0.001. (d) Representative confocal micrographs and
quantification of the platelet/B-cell interaction in the liver of
ND, CD-HFD fed and CD-HFD fed treated with Asp-Clo mice (n=4 mice
per group) are shown. Sinusoids are presented in blue, platelets
are presented in green and B-cells are presented in red. Scale bar
represents 50 .mu.m. Platelet count alone and platelet adjacent to
B-cells quantification are shown in focus of view (FOV) (n=4 mice
per group). For visualization of intravascular events and to
increase image clarity, the transparency of the sinusoidal
rendering was set to 50%. See also Movie S3 and Movie S4. (e)
Representative confocal images from the liver of ND, CD-HFD fed
mice and also Asp-Clo treated CD-HFD fed mice for platelet/CD8' T
cells interaction in region of interest (ROI), the same mice
visualized for (d). Sinusoids are presented in blue, platelets are
presented in green and CD8' T cells are presented in red. Scale bar
represents 50 .mu.m. Single platelet count as well as platelet/CD8'
T cell quantification are shown in focus of view (FOV) (the same
mice used for (d). For a-e, a two-tailed unpaired Student's t-test
was used for statistical analysis: *P<0.05, **P<0.01,
***P<0.001.
EXAMPLES
Example 1: The First Studied Mouse Model for Human NASH and
Subsequent HCC Development
[0055] Indeed that approach enabled the inventors to study a
chronic model of NASH in the context of metabolic syndrome,
triggering subsequent HCC in C57BL/6 mice, in the absence of
chemical carcinogens or genetic mutations predisposing to NASH or
HCC development. CD-HFD fed mice display several long term
pathologies observed in human patients: abdominal obesity,
overweight, insulin resistance, liver damage, production of
reactive oxygen species (ROS) fibrosis, hepatic mitochondrial
damage, dyslipidemia and NASH. Moreover, HCC developed 12 months
post CD-HFD start and resembled histologically, genetically and
morphologically human HCC (FIG. 1). Cholesterol Diet--High Fat Diet
(CD-HFD) in mice recapitulates human NASH and NASH triggered HCC
pathology. (FIG. 1A) Weight development in male normal diet fed
(ND) and CD-HFD fed C57BL/6 mice. (FIG. 1B) Intraperitoneal Glucose
tolerance test performed with 6-month-old male ND and CD-HFD
C57BL/6 mice. (FIG. 1C) Quantification of serum aminotransferase
(ALT) levels in male C57BL/6 mice reflecting liver damage. (FIG.
1D) MRI analyses on livers of 6-month-old ND and CD-HFD C57BL/6
mice. T1 (fast low-angle shot [FLASH]) OUT phase: dark color
indicative of steatosis. T2 TurboRare visualizes increase in
subcutaneous and abdominal fat and hepatic lipid accumulation
(bright regions) in CD-HFD mice. (FIG. 1E) Macroscopy and
histopathology of livers from 12-month-old ND, HFD, or CD-HFD
C57BL/6 mice, with arrowhead pointing towards HCC. Scale bar: 5 mm.
T: Tumor. NT: Non-Tumor. ***P=0.001. (F) Representative H&E
staining of 12-month-old CD-HFD C57BL/6 livers and human livers
illustrating NASH. Accumulation of Mallory Denk bodies (red
arrowhead), ballooned hepatocytes (brown arrowhead), and
satellitosis (blue arrowhead) is similar to human NASH pathology
(right image). Scale bar: 50 .mu.m.
[0056] The present mouse model, which recapitulates several
pathophysiological aspects of human NASH, provides the basis for
this invention and allows us to study the biology and development
of NASH and NASH to HCC transition. Notably, using this mouse model
it is demonstrated for the first time that CD8+ T-cells and
NKT-cells become activated during metabolic syndrome, interact with
hepatocytes via cytokines and alter hepatic lipid metabolism
causing NASH and HCC. An identical profile of CD8+ and NKT cell
activation was found in human NASH livers underlining the clinical
relevance of our model (Wolf et al., Cancer Cell, 2014).
[0057] Opposing results have been published in the past in the
frame of short-term in vivo experiments on the role of immune cells
in experimentally induced NASH (Martin-Murphy et al., 2014; Lynch
et al., 2012; Bhattacharjee et al., 2014). Notably, these models
lacked a metabolic syndrome and were usually kept on a diet for
only several weeks. In contrast, the present data, which are based
on a long-term CD-HFD leading to obesity, metabolic syndrome and
HCC in C57BL/6 mice, demonstrate that immune cells play an
important role in triggering steatosis, NASH and NASH-driven HCC
(Wolf et al., Cancer Cell, 2014). Moreover, the inventors could
demonstrate that the inflammation profile of livers from CD-HFD
mice looked similar to that of human NASH livers, e.g. as far as
inflammatory cells in the liver or cytokine expression is concerned
(FIG. 2).
[0058] In FIG. 2 from left to right: H&E, B220, CD3, F4/80,
MHCII, and Ly6G. Inflammatory aggregates consisting of CD3+, F480+,
and MHCII+ cells were observed. Scale bar: 50 .mu.m. (B) CD8+ T and
NKT cells control liver steatosis development. Sudan red staining
of liver sections (12-month-old, indicated genotypes) demonstrated
that depletion of B- and T-cells (Rag1-/- mice) or more
specifically CD8+ and NKT cells (.beta.2m-/- mice) sufficed to
strongly reduce the development of steatosis upon CD-HFD. In line a
reduction of other parameters such as aminotransferases, liver
triglycerides is found (not shown). Scale bar: 50 .mu.m. (C)
Representative IHC of human non-diseased control livers and livers
of NASH patients for CD8+ T cells, CD3+CD57+ NKT cells, LT.beta.
and LIGHT expression with arrowheads indicating positive cells
Scale bar: 50 .mu.m. Densitometric analysis of immune cells and
LIGHT expression analysed on mRNA level derived from human
cryomaterial.
Example 2: Platelets are Increased, Activated and Aggregate in
NASH
[0059] FIG. 3 shows an increase of platelets and aggregation as a
sign of activation in the liver of mice suffering from NASH upon 6
months CD-HFD. (FIG. 3A) Immunohistochemical images which identify
platelets to aggregate and to be increased in number. Platelets are
stained by GPIb. (FIG. 3B) Densitometric analysis of platelets show
a strong and significant increase in number in the livers of CD-HFD
treated mice. DATA NOT SHOWN: Similar images and a similar increase
in GPIb numbers can be seen in patients suffering from NASH when
compared to healthy patients.
[0060] FIG. 4 shows an analysis of activation and accumulation
capacity of platelets analysed by flow cytometry from peripheral
blood. Flow cytometry analysis indicates that there is a
significantly increased capacity of platelets to be activated
(upper) and a tendency to aggregate (lower). By using different
dilution curves of CRP or Thr a reproducible and consistent
analysis of the platelet-response rate was visible.
[0061] Aspirin/Clopidrogrel (Asp/Clop), a thrombocyte aggregation
inhibitor, treatment reduces the capacity of platelets aggregate
and prevents NASH and aberrant hepatic lipid metabolism (FIG. 5).
(FIG. 5A) Asprin/Clopidrogrel treatment reduces the number of
platelets in the liver (White bars: ND; Black bars: CD-HFD; Green
bars: CD-HFD treated with Asp/Clop). (FIG. 5B) Activation status of
platelets (investigated by an ex vivo assay) is significantly
reduced upon Asp/Clop treatment on platelets taken from peripheral
blood. (FIG. 5C) Aggregation capacity status (investigated by an ex
vivo aggregation assay) of platelets is significantly reduced upon
Asp/Clop treatment on platelets taken from peripheral blood. (FIG.
5D) Asp/Clop CD-HFD fed mice do develop obesity as CD-HFD mice.
(FIG. 5E) Liver to body weight ratio is rescued upon Asp/Clop
treatment at 12 months of age. (FIG. 5F) Liver damage as measured
by ALT levels in serum is prevented in Asp/Clop treated mice. (FIG.
5G) Triglycerids are reduced in serum of Asp/Clop treated mice at
12 months of age. (FIG. 5H) Serum cholesterol is reduced in
Asp/Clop treated mice at 6 and 12 months, (FIG. 5I) Aberrant
hepatic lipid metabolism and b-Oxidation is rescued in Asp/Clop
treated mice--as anaylzed by Real time PCR analysis for gene
expression in the liver. Stars indicate significance. (FIGS. 5J and
K) MRI analysis and histological analysis demonstrate that Asp/Clop
treatment prevents the development of NASH. (FIG. 5J) Lipid
deposition in the liver indicated by a dark staining in the T1
(FLASH) OUT phase is not detected--as in the CD-HFD. Still at the
same time abdominal and subcutaneous fat are visible in Asp/Clop
treated mice. (FIG. 5K) Histopathological signs of NASH (e.g.
balooned hepatocytes--see also stars) are absent in Asp/Clop
treated mice.
[0062] Furthermore, a reduced amount of intrahepatic immune cells
in Aps/Clp treated mice was observed (FIG. 6). (FIG. 6A) Reduction
of several immune cell types including CD3+ T cells, F480+ cells
and MHCII expressing cells. B220+ immune cells are not altered.
Moreover, (FIG. 6B) flow cytometry analysis shows a significant
reduction in the number of CD8+ T cells, the activation of
CD44+CD8+ T cells, CD69+CD8+ T cells as well as in the number of
CD3+NK1.1+ NKT cells (White bars: ND; Black bars: CD-HFD; Green
bars: CD-HFD treated with Asp/Clop).
Example 3: Inhibiting the Activity/Aggregation of Platelets Reduces
the Number of HCC
[0063] In the mouse model for NASH, the treatment with Asp/Clop
resulted in the reduction in HCC incidence (FIG. 7). Significant
reduction in the number of HCC in Asp/Clop treated mice. (White
circles: ND; Black circles: CD-HFD; Green circles: CD-HFD treated
with Asp/Clop low dose; red circles: Asp/Clop high dose).
Example 4: COX1, COX2 is not Involved in NASH Development
[0064] Sulindac, a COX1, COX2 inhibitor does not block steatosis or
NASH development upon CD-HFD (FIG. 8). (FIG. 8A) CD-HFD fed mice do
develop obesity as sulindac CD-HFD mice (White bars: ND; Black
bars: CD-HFD; Red bars: CD-HFD treated with Sulindac). (FIG. 8B)
Liver to body weight ratio is not rescued upon sulindac treatment
at 12 months of age. (FIG. 8C) Liver damage as measured by ALT
levels in serum is not prevented in sulindac treated mice. (FIG.
8D) Triglycerides are not significantly reduced in serum of
sulindac treated mice at 6 or 12 months of age. (FIG. 8E) Serum
cholersterol is not significantly reduced in Asp/Clop treated mice
at 6 and 12 months, (FIG. 8F) Sulindac treated, CD-HFD fed mice are
insulin resistant as CD-HFD fed mice, as investigated by an
intraperitoneal glucose tolerance test. (FIGS. 8G and H) MRI
analysis and histological analysis demonstrate that Sulindac
treatment does not prevent the development of NASH. (FIG. 8G) Lipid
deposition in the liver indicated by a dark staining in the T1
(FLASH) OUT phase is detected--as in the CD-HFD. At the same time
abdominal and subcutaneous fat are visible in sulindac treated,
CD-HFD fed mice as in CD-HFD fed mice alone. (FIG. 8H)
Histopathological signs as indicated by H/E stains of NASH (e.g.
balooned hepatocytes--see also stars) are present in sulindac
treated, CD-HFD fed mice. (FIG. 8I) Aberrant hepatic lipid
metabolism and b-Oxidation is not rescued in sulindac treated
mice--as analyzed by Real time PCR analysis for gene expression in
the liver. Stars indicate significance. (FIG. 8J) Lipid deposition
(as indicated by Sudan red) is not reduced in sulindac treated
CD-HFD mice at 12 months, as indicated by histological and
densitometric analysis.
Example 5: The Platelet Aggregation Inhibitor Ticagrelor Inhibits
NASH and HCC Development
[0065] Ticagrelor blocks NASH and HCC development (FIG. 9). (FIG.
9A) CD-HFD fed mice do develop obesity as ticagrelor treated CD-HFD
mice (White bars: ND; Black bars: CD-HFD; Green bars: CD-HFD
treated with ticagrelor). (FIG. 9B) Liver damage as measured by ALT
levels in serum is significantly reduced in ticagrelor treated
mice. (FIG. 9C) Triglycerides are reduced by trend in serum of
ticagrelor treated mice at 12 months of age. (FIG. 9D) Serum
cholersterol is significantly reduced in ticagrelor treated mice at
6 and 12 months. (FIG. 9E) Aberrant hepatic lipid metabolism and
b-Oxidation is rescued in ticagrelor treated mice--as analyzed by
Real time PCR analysis for gene expression in the liver. Stars
indicate significance. (FIG. 9F) Histopathological signs as
indicated by H/E stains of NASH (e.g. balooned hepatocytes--see
also stars) are not present in ticagrelor treated, CD-HFD fed mice.
(FIG. 9G) Lipid deposition (as indicated by Sudan red) is reduced
in sulindac treated CD-HFD mice at 12 months, as indicated by
histological and densitometric analysis. (FIG. 9H) No HCC in
ticagrelor treated mice.
Example 6: Nbeal2 Knock-Out Inhibits NASH and Prevents HCC
[0066] Nbeal2-/- mice lack development of markers indicative of
NASH development (FIG. 10). (FIG. 10A) CD-HFD fed C57BL/6 mice do
develop obesity as CD-HFD fed Nbeal2-/- mice (White bars: ND; Black
bars: CD-HFD; Blue bars: Nbeal2-/- mice on CD-HFD). (FIG. 10B)
Liver damage as measured by ALT levels in serum is significantly
reduced in CD-HFD fed Nbeal2-/- mice. (FIG. 10C) Triglycerids are
significantly reduced in serum of CD-HFD fed Nbeal2-/- mice at 6
months of age. (FIG. 10D) Serum cholersterol is significantly
reduced in CD-HFD fed Nbeal2-/- mice at 6 months. (FIG. 10E) CD-HFD
fed Nbeal2-/- mice are insulin resistant as CD-HFD fed mice, as
investigated by an intraperitoneal glucose tolerance test. (FIG.
10F) Aberrant hepatic lipid metabolism and b-Oxidation is partially
rescued in CD-HFD fed Nbeal2-/- mice--as analyzed by Real time PCR
analysis for gene expression in the liver. Stars indicate
significance.
Example 7: Gp1b Knock-Out Inhibits NASH and Prevents HCC
[0067] GP1b-/- mice lack steatosis and NASH development (FIG. 11).
(FIG. 11A) CD-HFD fed C57BL/6 mice do develop obesity as CD-HFD fed
Gp1b-/- mice (White bars: ND; Black bars: CD-HFD; yellow bars:
Gp1b-/- mice on CD-HFD). (FIG. 11B) Liver damage as measured by ALT
levels in serum is significantly reduced in CD-HFD fed Gp1b-/-
mice. (FIG. 11C) Triglycerides are strongly reduced in serum of
CD-HFD fed Gp1b-/- mice at 6 months of age. (FIG. 11D) Serum
cholersterol is significantly reduced in CD-HFD fed Nbeal2-/- mice
at 6 months. (FIG. 11E) Aberrant hepatic lipid metabolism and
b-Oxidation is partially rescued in CD-HFD fed Gp1b-/- mice--as
analyzed by Real time PCR analysis for gene expression in the
liver. Stars indicate significance. (FIGS. 11F and G) Steatotsis
and NASH (e.g. ballooned hepatocytes) are gone in Gp1b-/- mice.
Sudan red positive fatty droplets are quantified, corroborating our
data. (FIG. 11H) Activiation and aggragation capacity are
significantly reduced upon ticagrelor treatment.
Example 8: Anti-Platelet Treatment in Humans
[0068] FIG. 12A shows enzymes aspartate transaminase (AST) and
alanine transaminase (ALT) level of patients before study inclusion
and after 6 months of dual-anti platelet therapy (DAPT) with
Aspirin and Clopidogrel (n=148). FIG. 12 B shows an MRI and
sonography of patients on ASA or DAPT before and 6 months after
treatment. Quantification of liver fat deposition in human patients
as assessed by MRI is shown in FIG. 12C. The experiment shows a
significant effect of DAPT in the treatment of NASH.
Example 9: Anti Platelet Therapy Impairs Immune Cell Activity
[0069] Anti platelet therapy reduces chemokines and cytokines in
the liver, reduces ADP levels and prothrombin levels as shown in
FIG. 13 A-C. Further it was observed that antiplatelet therapy
reduces immune cells in the liver by blocking platelet immune cell
interaction (FIGS. 13 D and E).
* * * * *